Adenoviruses have been studied as a way to develop new treatments for different
diseases. Adenoviral vectors (AdV) are considered interesting tools for this propose,
because they can be produced at high titers (1X1012 particles per millilitre) in laboratory
and they have the capacity to infect non-dividing and dividing cells. AdV have been often
modified in order to obtain the ability to kill tumour cells or to deliver exogenous genetic
sequences essential to treat monogenic disease. However, weak expression of the primary
adenovirus receptor, the CAR (Coxsackie and adenovirus receptor) reduces greatly the
transduction efficiency of AdV for the tumour cells. Moreover, some normal tissues
express low amount of CAR, like the skeletal muscle, reducing the appeal of using AdV as
a gene delivery vehicle for this tissue. To address this problematic, many modifications
were done on the adenoviral capsid. The goal of these modifications were to generate an
AdV able to target specific cellular receptors that were expressed in tumour cells but not in
normal cells. Several approaches were done to modify the tropism of AdV, such as
incubation with a bispecific ligands, incorporation of peptides within the adenoviral fiber
structure or substitution of the viral fiber with a different serotype fiber.
The hypothesis of my project was to determine if an interaction domain fused within
a ligand could bind the complementary domain incorporated on a virus and change the
tropism of the AdV. The first step was to include a synthetic interaction domain, the K-Coil, within specific region of the adenoviral fiber, as well as inserting two point mutations
to abolish the natural tropism. To target the EGF-R, IGF-IR and the CEA6, we fused the
complementary interaction domain, the E-Coil, to the respective ligand known as the EGF
and the IGF-I or to a single domain antibody (known as AFAI) that bind specifically to
CEA6. The specific interaction between the E-Coil and K-Coil was used to associate the
ligand with the fiber in order to retarget the AdV toward the selected receptor.
We showed that the different ligands as well as the modified fibers could be
produced and that both E-Coil and K-Coil expressing partners could interact together. We
optimized the viral production by using an iodixanol purification protocol. More
importantly, we clearly demonstrated that the ligand association with the fiber could
increase the transduction efficiency between 2 to 21 fold against various tumour cells. The
difficulty of adenovirus to infect muscle cells because of the lack of CAR expression
brought us to evaluate the potential of our retargeted AdV to increase the transduction for
the tissue. We showed that the use of IGF-E5 could increase the transduction efficiency in
myoblasts as wells as in myotubes. We finally demonstrated that our retargeting system
could increase the transduction efficiency for skeletal muscle by 1,6 fold in new born MDX
mice. In conclusion, our results show that the retargeting system is indeed functional. This
system could be assessed using vectors that express therapeutic genes.